Specific incorporation of Se into polynucleotide structures of tRNAs has been observed in several bacterial species and mammalian cells. In each case, the incorporated Se is distributed among several tRNA species and at least two different selenonucleosides have been detected in Methanococcus vannielii. In Escherichia coli and Clostridium sticklandii the major selenonucleoside is 5-mnm-Se-2-U. In M. vannielii, another U modified seleno-base was observed in addition to 5-mnm-Se-2-U. Those seleno-tRNAs which contain 5-mnm-Se-2-U can be eluted from a RPC-5 colunn with 0.5 - 0.65 M NaCl, while those containing the other seleneo-base are eluted with much higher salt concentration (0.65 - 0.85 M). The glutamate accepting activities of tRNA preparations from C. sticklandii and M. vannielii are destroyed upon elimination of Se by CNBr treatment. Nuclease T1 digestion of the purified glutamate-accepting seleno-tRNA from C. sticklandii generated one major Se-containing oligonucleotide (25 bases long). Based on a comparison with E. coli glutamate-accepting tRNA, this seleno-oligonucleotide showed sequence homology from residues 26 to 50, including the anticodon region and the variable loop. Residue 34, which is the first nucleoside of the anticodon, is sensitive to CNBr treatment. These results suggest that 5-mnm-Se-2-U, like 5-mnm-S-2-U may be located in the "wobble" position of the anticodon. Selenium is regarded as an essential trace element for mammalian species and an important dietary anti-carcinogen against both chemical and virus induced tumors. It has been demonstrated that selenium can suppress the growth of mouse leukemia cells (L1210) in vivo. To elucidate the mode of action of selenium at the molecular level, selenium-containing tRNA species were isolated from cultured L1210 cells. The major seleno-tRNAs were eluted from an RPC-5 column at much higher salt concentration and the seleno-base has much higher hydrophobic character as compared to those of bacterial origin. Incubation with 15[3-H]-amino acids and E. coli aminoacyl-tRNA synthetase indicated that these seleno-tRNAs have some amino acid accepting activity.